In vivo neutralization of bee venom lethality by IgY antibodies.

Bee venom is a complex mixture of molecules, among which melittin and phospholipase A2 (PLA2) are the toxic components involved in envenoming accidents with multiple honeybee stings. Traditionally, the treatment of envenomings has been based on the administration of specific antibodies to neutralize the deleterious effects of toxins. An alternative to mammalian polyclonal antibodies is the use of egg yolk immunoglobulins (IgY) due to their advantages regarding animal welfare and lower costs of production as compared to the conventional production methods. In this work, a novel composition containing specific IgY antibodies was developed. After four immunizations, IgY extracted from the egg yolks was able to recognize several components of the bee venom, including melittin and PLA2. The performance of IgY to neutralize the lethal activity was evaluated in a mouse model by using one median lethal dose (LD50) of the bee venom. The effective dose of the IgY extract was determined as 30.66 µg/mg. These results demonstrate the feasibility to produce IgY-based antivenoms to treat envenomings by multiple bee stings.

Melittin-lipid nanoparticles target to lymph nodes and elicit a systemic anti-tumor immune response.

Targeted delivery of a nanovaccine loaded with a tumor antigen and adjuvant to the lymph nodes (LNs) is an attractive approach for improving cancer immunotherapy outcomes. However, the application of this technique is restricted by the paucity of suitable tumor-associated antigens (TAAs) and the sophisticated technology required to identify tumor neoantigens. Here, we demonstrate that a self-assembling melittin-lipid nanoparticle (α-melittin-NP) that is not loaded with extra tumor antigens promotes whole tumor antigen release in situ and results in the activation of antigen-presenting cells (APCs) in LNs. Compared with free melittin, α-melittin-NPs markedly enhance LN accumulation and activation of APCs, leading to a 3.6-fold increase in antigen-specific CD8+ T cell responses. Furthermore, in a bilateral flank B16F10 tumor model, primary and distant tumor growth are significantly inhibited by α-melittin-NPs, with an inhibition rate of 95% and 92%, respectively. Thus, α-melittin-NPs induce a systemic anti-tumor response serving as an effective LN-targeted whole-cell nanovaccine.

A biomimetic nanoparticle-enabled toxoid vaccine against melittin.

BACKGROUND: Melittin, the main active peptide ingredient of bee venom, can cause severe cell membrane lysis due to its robust interaction with negatively charged phospholipids. So far, no effective anti-melittin vaccine has been developed to protect people from undesired melittin intoxication. METHODS: Herein, we prepared a polydiacetylene (PDA) nanoparticle with cell membrane-mimic surface to complex melittin, forming an anti-melittin vaccine (PDA-melittin). RESULTS: PDA nanoparticles could effectively combine with melittin and neutralize its toxicity. PDA-melittin nanocomplex is demonstrated to enhance melittin uptake by DCs and stimulate strong melittin-specific immunity. Mice immunized with PDA-melittin nanocomplex showed higher survival rate after exposion to melittin than untreated mice. CONCLUSION: The PDA-melittin nanocomplex can efficiently and safely generate a specific immunity against melittin to protect body from melittin intoxication, providing a new method with potential clinical application for the treatment of melittin intoxication.

The protective effects of melittin on Propionibacterium acnes-induced inflammatory responses in vitro and in vivo.

Melittin is the main component in the venom of the honey bee (Apis mellifera). It has multiple effects including antibacterial, antiviral, and anti-inflammatory activities in various cell types. However, the anti-inflammatory mechanisms of melittin have not been elucidated in Propionibactierium acnes (P. acnes)-induced keratinocyte or inflammatory skin disease animal models. In this study, we examined the effects of melittin on the production of inflammatory cytokines in heat-killed P. acnes-induced HaCaT cells. Heat-killed P. acnes-treated keratinocytes increased the expression of pro-inflammatory cytokines and Toll-like receptor 2. However, melittin treatment significantly suppressed the expression of these cytokines through regulation of the NF-κB and MAPK signaling pathways. Subsequently, the living P. acnes (1 × 10(7) CFU) were intradermally injected into the ear of mice. Living P. acnes-injected ears showed cutaneous erythema, swelling, and granulomatous response at 24 hours after injection. However, melittin-treated ears showed markedly reduced swelling and granulomatous responses compared with ears injected with only living P. acnes. These results demonstrate the feasibility of applying melittin for the prevention of inflammatory skin diseases induced by P. acnes.

Bee venom phospholipase A2 induces a primary type 2 response that is dependent on the receptor ST2 and confers protective immunity.

Venoms consist of toxic components that are delivered to their victims via bites or stings. Venoms also represent a major class of allergens in humans. Phospholipase A2 (PLA2) is a conserved component of venoms from multiple species and is the major allergen in bee venom. Here we examined how bee venom PLA2 is sensed by the innate immune system and induces a type 2 immune response in mice. We found that bee venom PLA2 induced a T helper type 2 (Th2) cell-type response and group 2 innate lymphoid cell activation via the enzymatic cleavage of membrane phospholipids and release of interleukin-33. Furthermore, we showed that the IgE response to PLA2 could protect mice from future challenge with a near-lethal dose of PLA2. These data suggest that the innate immune system can detect the activity of a conserved component of venoms and induce a protective immune response against a venom toxin.

Role of the inflammasome in defense against venoms.

Venoms consist of a complex mixture of toxic components that are used by a variety of animal species for defense and predation. Envenomation of mammalian species leads to an acute inflammatory response and can lead to the development of IgE-dependent venom allergy. However, the mechanisms by which the innate immune system detects envenomation and initiates inflammatory and allergic responses to venoms remain largely unknown. Here we show that bee venom is detected by the NOD-like receptor family, pyrin domain-containing 3 inflammasome and can trigger activation of caspase-1 and the subsequent processing and unconventional secretion of the leaderless proinflammatory cytokine IL-1ß in macrophages. Whereas activation of the inflammasome by bee venom induces a caspase-1-dependent inflammatory response, characterized by recruitment of neutrophils to the site or envenomation, the inflammasome is dispensable for the allergic response to bee venom. Finally, we find that caspase-1-deficient mice are more susceptible to the noxious effects of bee and snake venoms, suggesting that a caspase-1-dependent immune response can protect against the damaging effects of envenomation.

Episodic hemorrhage during honeybee venom anaphylaxis: potential mechanisms.

Episodic hemorrhage is not a typical symptom of anaphylactic reaction to insect stings. Cases of reactions to honeybee (HB) sting or venom immunotherapy in which the uterus is the main target organ are very rare. Hemorrhage can be induced by HB venom components, especially melittin, which interfere with complement cleavage and bradykinin release. Both mechanisms are directly or indirectly associated with coagulation, thrombolysis, hemolysis, and smooth muscle tone. Induction of episodic hemorrhage through pathway destabilization in a defective bradykinin system or vulnerable organ may not be compensated by appropriate regulatory mechanisms. The pathological role of effectors is generally offset by the interaction of various regulatory systems, and the probability of hemorrhage is minimized thanks to this compensatory capability. In endometrial bleeding, the uterus becomes more vulnerable as a result of postmenstrual vascular fragility and additional induction of anaphylaxis-related uterine contractions. Episodic hemorrhage, especially metrorrhagia, as a consequence of HB venom activity may be suspected by an allergologist, but not by a physician. Melittin-free or recombinant allergens of HB venom, as well as modulators of the biochemical systems involved, could help to reduce the likelihood of hemorrhage. However, further investigation is required before these strategies can be introduced in clinical practice.

Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity.

The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations.

BACKGROUND: Treatment with aqueous and aluminum hydroxide (Al[OH](3))-adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. OBJECTIVE: The aim of the present study was to assess these purified HBV immunotherapy preparations in situ. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. RESULTS: Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH)(3)-adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. CONCLUSION: The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH)(3)-adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A(2)-induced degranulation in mast cells.

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of ß-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G(M1)), di-sialoganglioside (G(D1a)) and tri-sialoganglioside (G(T1b)). In contrast, honeybee venom-derived phospholipase A(2) induced the net degranulation directly without cytotoxicity, which was not inhibited by G(M1), G(D1a) and G(T1b). For analysis of distribution of Gα(q) and Gα(i) protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα(q) and Gα(i) at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A(2)-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A(2)-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

Recognition, neutralization, and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles: a plastic antibody.

We report that simple, synthetic organic polymer nanoparticles (NPs) can capture and clear a target peptide toxin in the bloodstream of living mice. The protein-sized polymer nanoparticles, with a binding affinity and selectivity comparable to those of natural antibodies, were prepared by combining a functional monomer optimization strategy with molecular-imprinting nanoparticle synthesis. As a result of binding and removal of melittin by NPs in vivo, the mortality and peripheral toxic symptoms due to melittin were significantly diminished. In vivo imaging of the polymer nanoparticles (or "plastic antibodies") established that the NPs accelerate clearance of the peptide from blood and accumulate in the liver. Coupled with their biocompatibility and nontoxic characteristics, plastic antibodies offer the potential for neutralizing a wide range of biomacromolecules in vivo.

Immune response to lytic peptides conjugated to a betaCG fragment in treated BALB/C mice.

Hecate-betaCG and Phor14-betaCG(ala) are relatively short, amphipathic alpha-helical cationic peptides with the ability to destroy selectively breast, prostate and ovarian cancer cells. Treatment with proteins and peptides frequently initiated antibody formation. Short peptides may minimize the risk of the immune system mobilization after treatment but it is necessary to investigate whether Hecate-betaCG and Phor14-betaCG(ala) induce the immune system to produce antibody and whether they affect the reproductive organs in normal wild-type mice. The results of our experiments showed that specific antibodies, tested by the enzyme-immunoassay, were not detected in the group treated with Hecate-betaCG and Phor14-betaCG(ala). The blood concentrations of both peptides begun to decrease from 60 minutes after injection and after 240 minutes its levels were undetectable. Histopatho-logical examination exhibited degenerative changes in the prostate glands and testes in males and in the ovaries and uteri of females treated with both peptides. In conclusion, our results indicate that both relatively small and rapidly metabolized peptides are not immunogenic and can be used for further investigation as a potential cancer treatment.

A protein whose binding to Na,K-ATPase is regulated by ouabain.

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.

Increased sensitivity of early apoptotic cells to complement-mediated lysis.

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

Adjuvant action of melittin following intranasal immunisation with tetanus and diphtheria toxoids.

Melittin, a 26-amino acid peptide and the major active component of the venom of the honey bee--Apis mellifera--has recently been shown to have absorption enhancing properties in Caco-2 cells at levels well below the level required for the generation of cytotoxicity. Given the potential of absorption enhancing agents to act as adjuvants when administered nasally [Alpar, H.O., Eyles, J.E., Williamson, E.D. and Somavarapu, S. (2001) "Intranasal vaccination against plague, tetanus and diphtheria", Adv. Drug Delivery Rev. 51, 173-201] we hypothesized that melittin may have potential as a mucosal adjuvant. Following our initial studies reported here, it was found that the co-administration of 4 microg of melittin in conjunction with tetanus toxoid in BALB/c mice was effective in eliciting markedly enhanced antibody titres in comparison to control groups and groups receiving free antigen administered intranasally. Lower concentrations of melittin were less effective and mice receiving 4 microg of melittin plus antigen exhibited antibody titres significantly higher (i.e. P<0.05) than any of the other groups tested. The observed IgG2a titres were shown to be dependent upon the dose of melittin co-administered with the immunising antigen in a similar fashion to the observed total IgG responses. In summary, melittin has been shown here to have potential as a novel mucosal adjuvant for antigens administered via the nasal route.

Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization.

In previous studies we have shown that lipopeptides constitute potent immunoadjuvants in mice, rabbits and other species: in parenteral immunization, lipopeptide adjuvants were comparable, or in some cases superior to Freund's adjuvant, and were devoid of the side effects of this additive. Here we demonstrate that lipopeptides also constitute adjuvants for mucosal immunizations. The serum antibody responses against the wheat storage protein gliadin, the bee venom constituent melittin, or the hen egg protein ovalbumin could in most cases be enhanced more than 100-fold by the lipopeptide P3CSK4, applied via the nasal route. An enhanced specific antibody level could also be detected in supernatants of cell cultures prepared from spleens, Peyer's patches, lungs and mesenteric lymph nodes of immunized mice. Moreover, the lipopeptide P3CSK4 enhanced chemiluminescence in mouse spleen cells and peritoneal macrophages in vitro, indicating a macrophage-activating effect. Finally, nasal application of lipopeptide increased protection against a lethal infection of influenza. Our findings are of importance for the improvement of immunizations and might lead to more effective vaccines.

Antibody responses to bee melittin (Api m 4) and hornet antigen 5 (Dol m 5) in mice treated with the dominant T-cell epitope peptides.

BACKGROUND: Mice treated with the dominant T-cell epitope peptides of allergens were reported to have reduced peptide or allergen-specific T-cell responses on subsequent immunization, but the extent of reduction of allergen-specific antibodies is not clear. OBJECTIVE: This study was done to compare the extent of reduction of T-cell and antibody responses in peptide-treated mice. Two allergens were tested. Bee melittin (Api m 4), an allergen of 26 amino acid residues, has a single dominant T- or B-cell epitope. Hornet antigen 5 (Dol m 5), an allergen of 204 amino acid residues, has multiple dominant T- or B-cell epitopes. METHODS: Mice were treated with T-cell peptides of Api m 4 or Dol m 5 and then immunized biweekly with their respective allergen with alum adjuvant. T-cell peptides tested were residues 7-19 of Api m 4 and residues 41-60, 141-160, and 176-195 of Dol m 5. T-cell responses at week 9 or 11 were assayed by proliferation of spleen cell cultures. Antibody responses of different isotypes were measured biweekly by ELISA. RESULTS: Partial reduction of 30% to 50% of T-cell responses to peptide or allergen was observed in bee and hornet peptide-treated mice. About 65% reduction of Api m 4-specific antibody response was observed early in the immune response but gradually subsided to about 40% late in the response. Partial reduction of about 40% of Dol m 5-specific antibody response was only observed early in the immune response. CONCLUSION: Peptide treatment is partially effective in the reduction of T-cell responses of univalent or multivalent allergens. It is also partially effective in the reduction of antibody response of a univalent allergen, but it is poorly effective for a multivalent allergen.

Molecular parameters in melittin immunogenicity.

Based on immunogenicity studies, two T-cell epitopes in melittin were found to be functional in guinea pigs, one being centrally located, the other one residing in the C-terminal chain. In Balb/c mice only the central epitope was found to be active. A human T-cell clone was found by T-cell proliferation studies to employ strictly the C-terminal chain. Truncation of melittin peptides at the N-terminus did not markedly affect the capacity of guinea pigs to develop anti-IgG responses towards peptidic epitopes and towards a C-terminally attached haptenic group. Attachment of various substituents inside and outside the T-cell epitopic areas had no marked effect on antibody responses. In contrast, the substituents positioned within a T-cell epitope abolished T-cell proliferation. This difference between whole animal data and cellular in vitro responses is presently not understood.